Overexpression of the integral membrane protein P-glycoprotein (P-gp, also known as the multidrug resistance (MDR) transporter) is the most common characteristic associated with tumor cells that have developed drug resistance to antineoplastic agents (Gottesman et al, Ann. Rev. Biochem. 62:385 (1993), Endicott et al, Ann. Rev. Biochem. 58:137 (1989)). In this type of drug resistant cell, P-gp functions as an energy-dependent drug efflux pump that can transport a broad spectrum of structurally unrelated cytotoxic and anticancer drugs.
P-glycoprotein is the product of the MDR1 gene. Homologous mammalian genes include the highly-related MDR2 gene and the MRP (for multidrug transporter related protein) gene (Gottesman et al, Ann. Rev. Biochem. 62:385 (1993); Cole et al, Science 258:1650 (1992)). To date, only the product of the MDR1 gene (ie P-glycoprotein) has been shown to be significantly associated with multidrug resistance. Additionally, the product of a related gene, pfmdr, in Plasmodium falciparum (the organism that causes malaria) has been implicated in chloroquine resistance in this parasite (Cowman et al, Seminars Cell Biol. 4:29 (1993)). Drug resistance in other parasites, eg trypanosomes, may also involve membrane transporters of the general structure of P-gp (Carter et al, Nature 361:173 (1993)).
P-glycoprotein belongs to a superfamily of membrane-associated transporters termed the ATP-binding cassette (ABC) family or the Traffic ATPase family (Higgins, Ann. Rev. Cell Biol. 8:67 (1992)). In mammalian cells, the best-known members of this family, in addition to P-gp, are the CFTR protein, the product of the cystic fibrosis gene (Welsh et al, Neuron 8:821 (1992)), a peroxisomal membrane protein termed PMP70 which has been implicated in the biogenesis of this organelle (Gartner et al, Seminars Cell Biol. 4:45 (1993)), and members of the family of adenylyl cyclases, the enzymes responsible for the production of cAMP as a second messenger (Tang et al, Cell 70:869 (1992)).
One such transporter similar to P-gp, termed Ste6, has been found in Saccharomyces cerevisiae to be responsible for the export of the peptide mating pheromone, a-factor (Michaelis, Seminars in Cell Biol. 4:17 (1993)). In a Ste6 deletion mutant, expression of a mammalian P-gp gene has been shown to complement the function of Ste6 and restore low, but detectable, mating activity (Raymond et al, Science 256:232 (1992)). Homology between P-gp and Ste6 thus appears to exist, from the standpoint of both function and structure.
Mature a-factor is a dodecapeptide containing a post-translationally modified C-terminus (Anderegg et al, J. Biol. Chem. 263:18236 (1988)). More specifically, the C-terminus of a-factor is subject to post-translational modification via the process of prenylation (Clarke, Ann. Rev. Biochem. 61:355 (1992)). Proteins subjected to this processing contain a cysteine residue at or near their C-terminus and are modified by attachment of either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid to the cysteine residue. The majority of prenylated proteins, which include Ras and most G proteins (Casey, Curr. Opinion Cell. Biol. 6:219 (1994)), are further modified by methylation on the carboxyl group of the prenylated cysteine.
Prenylation has been found to be a stable modification (Casey et al, Proc. Natl. Acad. Sci. USA 86:8323 (1989)) and prenylated proteins have been reported to comprise up to 2% of total cellular protein (Epstein et al, Proc. Natl. Acad. Sci. USA 88:9668 (1991)). These factors raise the question of how cells dispose of isoprenoid-modified cysteines produced during protein turnover. Effective disposal may be important as accumulation of such metabolites in cells could result in critical problems due to the fact that certain prenylcysteines can inhibit cellular processes such as protein methylation and signal transduction (Hazoor-Akbar et al, Proc. Natl. Acad. Sci. USA 90:868 (1993), Philips et al, Science 259:977 (1993)).
Prenylcysteine methyl esters, corresponding to the C-terminus of prenylated proteins, have now been identified as substrates for P-gp. This finding has provided an answer to the question of how the cell rids itself of potentially toxic metabolites and has provided basis for the present invention.